The practice of HPLC generally requires that a molecular species to be separated or analyzed be dissolved in a liquid, the mobile phase, and conveyed by that liquid through a stationary phase. In the stationary phase, a large surface area is presented which is in intimate contact with the mobile phase. Mixtures of analyte compounds, dissolved in the mobile phase, can be separated during passage through the column by processes of adsorption or retention, which act differently on the various analyte species. The differential retention causes the analytes to elute from the column with respect to time and volume. The eluting analytes will typically transit through an in-line detector, where quantitative and/or qualitative examination of analytes will occur.
High pressure liquid chromatography (HPLC) solvent delivery systems are used to source either single-component liquids or mixtures of liquids at selected pressures which can range from substantially atmospheric pressure to pressures on the order of ten thousand pounds per square inch. The above pressures are required to force the mobile phase through the fluid passageways of a stationary phase support, where separation of dissolved analytes can occur. The stationary phase support may comprise a packed bed of particles, a membrane or collection of membranes, a microfabricated structure typically comprising an array of fluid passageways etched into a solid support, or an open column or tube.
Often, analysis conditions require the mobile phase composition to change over the course of analysis which is termed "gradient elution". In gradient elution, the viscosity of the mobile phase may change and the pressure necessary to maintain the required volumetric flow rate will change accordingly.
The elution behavior of analyte molecules is a function of the characteristics of both the stationary and mobile phases. To the extent that the properties of the stationary phase may remain substantially fixed throughout the analysis, variation in elution behavior is predominantly a result of variation in the properties of the mobile phase. In an "isocratic mode" of chromatography, the solvent composition remains substantially constant as a function of time, and analytes in the sample will tend to elute when a prescribed mobile phase volume has transited the column. In a "gradient mode" of chromatography, the solvent composition is required to change as a function of time, tracking a user defined profile. In this mode, analytes will elute in response to both the composition and the volume of solvent delivered.
The separation process occurring in liquid chromatography can result in the separation of an injected sample mixture into its component parts. These component parts are eluted from the column in reasonably distinct zones or bands. As these bands pass through a detector, their presence can be monitored and a detector output can be produced. This output includes a pattern of analyte concentration within the eluting bands, which can be represented by means of a time varying electric signal, and gives rise to the nomenclature of a "chromatography peak." These peaks may be characterized with respect to their retention time, that is, the time in which the center of the band transits the detector relative to the time of injection. In many applications, the retention time of a peak is used to infer the identity of the eluting analyte based upon related analyses incorporating standards or calibrants. The retention time of a peak is strongly influenced by the mobile phase composition and by the volume of mobile phase, which has passed over the stationary phase.
The utility of chromatography relies heavily on run-to-run reproducibility, such that a given analysis can be compared with an analysis of standards or calibrants with confidence in the resulting data. Known pumping systems exhibit some non-ideal characteristics which result in diminished separation performance and diminished run-to-run reproducibility.
Among the non-ideal pump characteristics exhibited in known pumping systems are, generally, fluctuations in solvent composition and/or fluctuations in volumetric flow rate. Such volumetric flow rate fluctuations in present and known HPLC pumping systems disadvantageously cause varying retention times for a given analyte. That is, the amount of time that an analyte is retained in the stationary phase fluctuates undesirably as a function of the undesirable volumetric flow rate fluctuations. This creates difficulties in inferring the identity of a sample from the retention behavior of the components. Volumetric flow rate fluctuations can result in fluctuations in solvent composition when the output of multiple pumps is summed to provide a solvent composition.
Fluctuations in solvent composition in present and known HPLC systems disadvantageously result in interactions with the systems analyte detector and produce perturbations which are detected as if they arose from the presence of a sample. In effect, an interfering signal is generated. This interfering signal is summed with the actual signal attributable to the analyte, producing errors when the quantity of an unknown sample is calculated from the area of the eluting sample peak.
In light of the above, the requirements imposed on HPLC solvent delivery systems are severe. HPLC pumps are typically required to deliver solvents at pressures which can range from several pounds per square inch to as much as 10,000 pounds per square inch. There are problems and non-ideal effects associated with delivering liquids for chromatography against elevated pressures including seal deformation under load and absolute seal leakage. HPLC pumps are expected to output the mobile phase solvent at precisely controlled flow rates in a smooth and uniform manner. In the case of gradient chromatography or in the case of isocratic chromatography, where a fixed solvent composition is blended in real time during the separation, there is the further requirement that mobile phase composition as well as flow rate be precisely and accurately controlled during delivery. However, system operating pressures may be changing very substantially during the separation and the compressibilities of the constituent mobile phase solvents may be quite different.
The large errors associated with the compression or relaxation of large volumes of fluid can be minimized by the use of small volume syringe pumps that utilize multiple syringe strokes to deliver solvent through the course of a chromatographic separation period. However, these pumps typically suffer from flow perturbations associated with the transition of fluid delivery from one syringe cycle to the next.
An emerging area of chromatographic separation and analysis is developing around the use of extremely narrow bore separation columns. Such columns have been termed "capillary columns" with diameters typically in the range of 30 to 800 micron internal diameter. Such columns may be packed with a particulate packing material, or in the smallest diametral range, the stationary phase may be provided by the column wall itself or a coating applied to that wall. Mobile phase flow rates for such particulate packed capillary columns can typically range from approximately one nanoliter per minute to ten or more microliters per minute. These figures represent a three to six order-of-magnitude reduction in flow rate and consequently a similar reduction in the volume of the separation from what is currently practiced on, for example, the four millimeter internal diameter columns widely commercially available. HPLC systems designed around capillary columns have particular utility when the HPLC separation is coupled with a downstream process that does not readily tolerate large amounts of HPLC mobile phase, or where the use of unusually expensive mobile phases is desired. Examples of such processes are: (1) mass spectrometry, which requires that the sample reside in the gas phase at high vacuum conditions prior to mass analysis, (2) infrared spectroscopy, where organic solvents used for HPLC must be eliminated because they represent an interference to analyte detection in the infrared region of the electromagnetic spectrum, (3) microfraction collection, which requires that the analyte be deposited in a small volume on a collection substrate with minimum associated background contamination from the HPLC mobile phase, and (4) nuclear magnetic resonance spectroscopy (NMR) which can benefit from significant signal background reduction through the use of somewhat exotic mobile phases, such as deuterium-substituted mobile phases in the case of proton-NMR.
Substantially, the same requirements for precision and accuracy of solvent composition and flow rate delivery exist as for larger scale chromatography, but the mechanisms for controlling delivery must function at approximately one one-thousandth or less of the conventional volume scale. In particular, the non-idealities of a given implementation which could be dismissed at a much larger volumetric scale give rise to overwhelmingly large perturbations to a system of the scale of capillary HPLC.
A known implementation of a continuous-delivery pumping system for normal analytical scale HPLC (1 to 4 mm column ID), available from Hewlett-Packard and selling under the product designation HP1090/DR5, formed a gradient at run time using a plurality of positive-displacement piston pumps operating against a modest intermediate pressure. The summed output was fed to a low-pressure accumulator and then to a serial or in-line booster pump stage constructed on a diaphragm principle. The booster pump operated at a fixed frequency of approximately 10 Hz, but had a variable stroke volume which accommodated the output flow of the solvent metering/proportioning stage. The booster stage would take the proportioned mixture from the intermediate pressure to the full system operating pressure, which was in the conventional (5000 to 6000 PSIG) HPLC range. Because of the series construction employed, the pump had a delay volume of not less than approximately 300 microliters. This delay volume is quite acceptable for 2 mm ID and 4 mm ID column work, but becomes significant at 1 mm ID column size, and is inappropriate for work in the capillary realm, particularly so when the capillaries extend down to 30 to 50 micron internal diameter, and the flow rates are in the nanoliters-per-minute region.
A further disadvantage of that implementation is that the diaphragm booster stage imparted a significant AC pressure pulsation in the range of 100 PSIG, at the 10 Hz stroke frequency, to the downstream system. The existence of this AC pulsation in turn required the use of a compliant pulse damper in continuity with the diaphragm pump output stream, which is not desirable from several standpoints. Finally, because the system was designed to perform continuous solvent delivery at flow rates up to 5 milliliters per minute, as required for 4 mm ID columns, each of the intermediate-pressure solvent pumps was itself a continuous-delivery reciprocating pump comprising two cylinders and associated valving, resulting in a complex and expensive system.
Davis, M. T., et al., J. Am Soc. Mass Spectrom., 6, 571-577 (1995) describe an alternate implementation of a solvent delivery system designed to be operable at a capillary size scale. The approach disclosed by Davis et al. involves forming gradients by employing two low-pressure syringe pumps to generate a solvent composition which temporarily resides in a loop of open tubing. This loop is connected at each end to a commercially available six-port switching valve, following the manner of an external loop injector. Upon actuation of the switching valve, the fluid contents of the external loop are placed in continuity with the column, and with a high-pressure-capable syringe pump. The syringe pump is operated as a pressure source to expel the fluid contents of the external loop through the capillary analytical column. In arguing for a constant-pressure mode of operation, the authors acknowledge that their system is substantially incapable of constant-flow operation, as "There is no direct measurement of the actual flow from the column, and no feedback to the pump to maintain that flow rate. The piston is simply moved at a constant rate, and it can take a surprisingly long time for a system to come to equilibrium, and measured flow rates can change considerably during the course of a run." The system which Davis et al. describe includes a very large-volume syringe, representing a significant hydraulic capacitance, connected to a relatively small-diameter, and therefore substantially restrictive, capillary column. Such a system would indeed be expected to suffer from a very long hydraulic time-constant, or flow-rate settling time, in response to either a resistance change of the analytical column, or a flow-rate change at the syringe pump. This settling time could range from many minutes to hours. Therefore, for all practical purposes, during the course of a chromatographic run, there would be little correlation expected between the syringe pump delivery flow rate and the column flow rate. The authors claim that "there is no significant difference between constant pressure and constant flow operation when peptides and proteins are separated by capillary HPLC", yet acknowledge that during constant pressure operation, there are flow rate changes arising from the changes in solvent viscosity during gradient elution, and that constant-pressure operation is sensitive to "flow problems caused by sample precipitation or other blockage in the tubing or on the column".
In fact, when samples drawn from biological matrices are separated by capillary chromatography, it is not unusual to see the column resistance change measurably from one run to the next. This effect is superimposed upon the column resistance variation typically observed from one column to another, which is attributable to minor variations in physical construction and packing. Even small flow rate changes impair the user's ability to quantitate sample amounts from detector peak areas, since the area measurement assumes a constant flow rate. Larger flow rate changes impact system properties such as the ability to successfully maintain stable spray behavior in electrospray/nanospray interfaces. These examples illustrate why HPLC has been optimally practiced at constant volumetric flow rate as opposed to constant pressure. The Davis system provides the user with substantially no quantitative measure of the actual delivery flow rate provided to the column.
Finally, the approach disclosed by Davis et al. teaches the use of a loop of open tube as a gradient storage device. FIG. 3 in the Davis reference shows that there is only a very crude relationship between the called-for and the actual gradient composition profile delivered to the analytical column when a loop of open tube is used as the storage device. This behavior is not surprising in light of the fact that an open tube storage device will be susceptible to compositional band or zone broadening due to the Poiseuille flow profile attained in the open tube during fluid loading and expulsion, and to density-driven fluid motion while the solvent composition gradient is resident in the tube.
Recently, interest has developed in the ability to perform BPLC separations using extremely small diameter packing materials, that is, less than three micron diameter, with concomitantly high mobile phase pressure, which is required to drive the liquid through a bed filled with such packing particles. Enhanced separation characteristics of HPLC are demonstrated, in particular, either the absolute peak capacity of the separation, or the throughput of the separation as expressed in peaks eluted per unit time, can be substantially improved through the correct use of small packing particles and a high system pressure. The utility of particles as small as one micron diameter and system pressures in the rage of 10,000 to 100,000 PSIG has been demonstrated. However, in such analysis, power dissipation in the form of self-heating of the columns can be significant under the conditions of extremely high pressure drop over the length of the column. Therefore, it is desirable to maintain the column diameter as small as is feasible, often in the range of 30 to 75 micron internal diameter. These small column diameters, with correspondingly small volumetric flow rate requirements, help maintain the total heat generated to a relatively small value, and also keep the thermal paths for heat dissipation relatively short.
In using such small diameter columns (30 to 75 micron internal diameter), the actual volume of separation becomes exceedingly small, given that the mobile phase flow rates are typically in the 5 to 200 nanoliter per minute range. Gradient formation requires that the individual components of the mobile phase must be delivered at levels as low as 0.1% of the total system flow rate. In addition, high-pressure gradient formation pumps must deliver the component flows in a manner which is reproducible in fashion, against the full system operating pressure, without perturbations. The above described analytical requirements imply the ability to quantitatively deliver component flow rates as small as 10 picoliters per minute against pressures as high as 100,000 PSIG. Known HPLC systems do not provide the sealing and actuator technology required for the above stated requirements at a cost which is reasonable in the intended market.
Therefore, in light of the above, flow-splitting based systems have come under investigation for performing separations using extremely small packing particles with concomitantly high mobile phase pressure to drive the fluid through the bed of packing particles. These flow-splitting systems use solvent composition generation and pressure augmentation at a size scale of convenience. This larger scale flow is directed to a shunt path which includes significant resistance in line. The small scale gradient flow required for the above-stated 30 to 75 micron internal diameter columns is tapped from the larger scale flow by means of a tee and is subsequently conveyed through the injection means toward the column. Such systems have been employed extensively in capillary chromatography at modest pressures (0 to 5,000 PSIG) and their operation is being explored at more elevated pressures in the range of 6,000 to 20,000 PSIG. Drawbacks of these flow-splitting systems become apparent when system pressures reach the 50,000 to 100,000 PSIG levels, in part due to heat dissipation in the shunt path. In extreme cases, the heat generated is enough to convert the prescribed liquid into a vapor, or at a minimum, a liquid having a substantial vapor component.
Thus it should be apparent that the implementation of systems intended for use with extremely small packing particles and high mobile phase pressures involves severe design challenges.